System and method for obtaining a cellular sample enriched with defined cells such as platelet rich plasma (prp)

ABSTRACT

A system for obtaining plasma enriched in platelets is disclosed which is closed to the atmosphere. The system includes: a collection tube containing an anticoagulant portion and a separation gel; a first collection syringe adapted to collect a portion of fluid relatively depleted in platelets from said collection tube after centrifugation; and a second collection syringe adapted to collect plasma enriched in platelets from said collection tube after centrifugation, said second collection syringe further comprising a filter unit adapted to filter cells included in said plasma enriched in platelets.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 14/373,978, filed Jul. 23, 2014, which is a U.S. national phase of PCT Application Number PCT/IL2013/050061, filed on Jan. 23, 2013, which claims priority from provisional application No. 61/589,401, filed on Jan. 23, 2012, all of which are incorporated by reference.

FIELD OF THE INVENTION

Various embodiments of the invention relate to systems and methods for preparing a cellular fraction enriched with defined cells reported to be effective in promoting a defined cellular response from an in vivo cellular sample and more particularly systems and methods for preparing a platelet-rich plasma (PRP), enriched mesenchymal stem cells (MSC) or enriched bone marrow stem cells (BMSC) sample.

BACKGROUND OF THE INVENTION

The bone tissue is composed of bone matrix and bone cells. Bone matrix comprises mainly type I collagen as well as other proteins, including osteocalcin and osteonectin. These and other proteins take part in matrix maturation, mineralization and may regulate the functional activity of bone cells, the latter being primarily osteoblasts and osteoclasts. Osteoblasts are bone-forming cells and osteoclasts are bone resorbing cells. The main function of the bone cells is to mediate bone remodeling mechanisms in order to maintain the integrity of the skeleton by removing old bone of high mineral density and high prevalence of fatigue microfractures through repetitive cycles of bone resorption and bone formation. During bone formation phase, the osteoblasts are recruited from stem cells population present in bone marrow.

There are two main populations of stem cells in the bone marrow: hematopoietic stem cells (HSC), which give rise to all blood cell lineages including erythrocytes, platelets, and white blood cells), and mesenchymal stem cells (MSC). The MSC are self-renewing, clonal and multipotent precursors of nonhematopoietic tissues, capable of differentiating into osteoblasts, chondrocytes, astrocytes, pneumocytes, hepatocytes, neurons, and cardiac myocytes.

Bone marrow-derived stem cells (BMSCs) contribute to tissue repair or regeneration of a broad spectrum of tissues including myocardium, cardiac valves, blood vessels, damaged bone, simple bone cysts, and fracture delayed union or non-union tendon, cartilage, 2 meniscus, and skin. Bone marrow aspiration can be obtained from any of the standard autograft locations, e.g., iliac crest, proximal/distal tibia, or calcaneus. Standard technique for BM aspiration is withdrawal of the later using a suitable needle, such as trocar or bone marrow biopsy needle and an anticoagulant-containing syringe.

PRP is a product of blood plasma that is rich in platelets. PRP may be used in a variety of therapeutic or cosmetic applications including enhancing wound healing in dental implants and sinus elevations, heart surgery, orthopedic surgery, and dermatology (chronic wound healing) [Barry L. et al, Plastic Reconstructive Surgery 114(6): pp. 1502-1508, 2004; Barry L. et al, Plastic Reconstructive Surgery 118(6): 147e-15, 2006; and Kajikawa Y. et al, J. Cell Physiol., 215(3): pp. 837-45, 2008; and Jacques Otto (published online: http://www.theottoclinic.ie/skin_rejuvenation.htm). PRP may also be utilized as a culture medium for cell expansion in the laboratory.

WO2005/065269 discloses several compositions comprising PRP and fibroblast cells for the treatment of skin, in particular, repeated administration of PRP in a dermatologically acceptable carrier to skin to e.g. reduce appearance of wrinkles.

Thus there is a long unmet need to provide methods and means to obtain PRP, MSC and BMSC, in a state ready for use in therapeutic or cosmetic applications or procedures.

SUMMARY OF INVENTION

It is an object of the present invention to disclose a system for obtaining cellular fraction enriched with defined cells effective in promoting a defined cellular response from an in vivo cellular sample comprising:

(a) a collection tube for collecting a cell suspension (1), (b) an anticoagulant portion (4); (c) a portion of separation gel (3); (d) a first collection syringe for collecting a cell depleted fluid (10); (e) a second collection syringe for collecting the enriched cellular portion (11); and (f) at least one needle (10) for attaching to each syringe.

The system is closable to the atmosphere; further wherein the cell suspension collection tube contains the gel (3) and the anticoagulant (4); the tube is adapted such that, when containing cell suspension and centrifuged after treatment yields separation fractions, a first fraction comprising cellular fluid (6), a second fraction comprises gel layer (3), a third fraction comprises enriched cellular portion (7) on top of the gel layer and a fourth fraction comprises a cell poor portion (8).

It is another object of the present invention to provide the system as defined above, wherein the system is adapted such that the cell poor portion is discardable with the first collection syringe with the needle.

It is another object of the present invention to provide the system as defined above, wherein the second collection syringe with the needle are dimensionally configured for extraction of cells remaining after re-suspending of the enriched cellular portion.

It is another object of the present invention to provide the system as defined above, wherein the cell suspension is a peripheral blood sample or stem cell-containing cellular sample derived from tissue such as bone marrow or adipose stem cells.

It is one object of the present invention to provide a system for obtaining a low medium density fraction enriched with defined cells effective in promoting a defined cellular response from an in vivo cellular sample comprising: (a) a collection tube for collecting a cell suspension (1); (b) an anticoagulant portion (4); (c) a portion of separation gel (3); (d) a first collection syringe for collecting a cell depleted fluid (10); (e) a second collection syringe for collecting the enriched cellular portion (11); and, (f) at least one needle for attaching to each syringe.

The system is closable to the atmosphere, further wherein the cell suspension collection tube contains the gel (3) and the anticoagulant (4); the tube is adapted such that, when containing cell suspension and centrifuged yields fractions of different densities, a first high density fraction (6), a second medium-high density fraction (3), a third medium-low density fraction (7) on top of the gel layer and a fourth fraction low density fraction (8), the third medium-low density fraction substantially containing enriched defined cells; the enriched defined cells selected from the group consisting of enriched bone marrow stem cells, enriched MSC, and platelet rich plasma (PRP).

It is one object of the present invention to provide a system for obtaining an enriched mesenchymal stem cell (MSC) portion comprising: (a) a collection tube for collecting cell suspension (1), (b) at least one stopper (2), (c) an anticoagulant portion (4), (d) a portion of separation gel (3), a first collection syringe for collecting poor cellular fluid (10), (d) a second collection syringe for collecting mesenchymal stem cells (MSC) (11), and (e) at least one needle (10) for attaching to each syringe.

The system is closable to the atmosphere; further wherein the collection tube comprising the gel (3) and the anticoagulant (4); the collection tube containing cell suspension is adapted such that, when containing the cell suspension (5) and centrifuged after treatment yields separation fractions, a first fraction comprises primarily red blood cells (RBCs), a second fraction comprises gel layer (3), a third fraction comprises cellular fraction containing MSC (7) on top of the gel layer and a fourth fraction comprises poor cellular fluid (8).

It is another object of the present invention to provide the system as defined above, wherein the system is adapted such that the poor cellular fluid is discardable with the first collection syringe.

It is another object of the present invention to provide the system as defined above, wherein the second collection syringe and the needle are dimensionally configured for extraction of the enriched MSC remaining after re-suspending of the MSC portion.

It is one object of the present invention to provide a system for obtaining an enriched bone marrow fraction comprising:

(a) a collection tube for collecting bone marrow, (b) at least one stopper; (c) an anticoagulant portion, (d) a portion of separation gel; (e) a first collection syringe for collecting poor cells fluid, (f) a second collection syringe for collecting enriched bone marrow cells and, (g) at least one needle for attaching to each syringe.

It is another object of the present invention to provide the system as defined above, wherein the system is closable to the atmosphere; further wherein the collection tube for collecting bone marrow contains the gel and the anticoagulant; the tube containing the bone marrow is adapted such that when centrifuged after treatment yields separation fractions, a first fraction comprises primarily red blood cells (RBCs) sediment, a second fraction comprises gel layer, a third fraction comprises enriched bone marrow cells on top of the gel layer and a fourth fraction comprises poor cellular fluid.

It is another object of the present invention to provide the system as defined above, wherein the poor cellular fluid is discarded using the first collection syringe.

It is another object of the present invention to provide the system as defined above, wherein the second collection syringe with the needle are dimensionally configured for extraction of the enriched bone marrow cells after re-suspending of the remaining bone marrow cells portion.

It is another object of the present invention to provide the system as defined above, further comprising means for treating cell aspirate before the latter is applied into the collection tube.

It is one object of the present invention to provide a system for obtaining platelet rich plasma (PRP) comprising: (a) a collection tube for collecting blood (1), (b) at least one stopper (2), (c) an anticoagulant portion (4), (d) a portion of separation gel (3), (e) a first collection syringe for collecting platelet poor plasma (PPP) (10), (f) a collection syringe for collecting platelets rich plasma (PRP) (11), and (g) at least one needle for attaching to each the syringe. The system is closable to the atmosphere; further wherein the collection tube comprising the gel (3) and the anticoagulant (4); the collection tube is adapted such that when containing the whole blood (5) and centrifuged after treatment yields separation fractions, a first fraction comprises red blood cells (RBCs) sediment and granulocytes (6), a second fraction comprises gel layer (3), a third fraction comprises platelets (7) on top of the gel layer, and a fourth fraction comprises platelet poor plasma (PPP) (8).

It is another object of the present invention to provide the system as defined above, wherein the PPP portion is discardable using a platelet poor plasma (PPP) collection syringe.

It is another object of the present invention to provide the system as defined above, wherein the collection syringe for collecting platelets rich plasma (PRP) with the needle are dimensionally configured for extraction of the remaining PRP after re-suspending of the PRP portion.

It is one object of the present invention to provide a system for obtaining platelet rich plasma (PRP) comprising:

(a) a collection tube for collecting blood (1), (b) at least one stopper (2), (c) an anticoagulant (4), (d) a separation gel (3), (e) a collection syringe for collecting platelet poor plasma (PPP) (10), (f) a collection syringe for collecting platelet rich plasma (PRP) portion (11); and (g) at least one needle for attaching to each syringe, wherein the system is closable to the atmosphere; further wherein the collection tube is a vacuum tube and the separation gel and the anticoagulant is in the tube; further wherein the collection syringe for collecting platelets rich plasma (PRP) portion further comprises a filter unit of 3-50 μm for obtaining WBC-selective PRP.

It is another object of the present invention to provide the system as defined above, further comprises a filter unit of 3-50 μm.

It is another object of the present invention to provide the system as defined above, wherein the collection tube is selected from the group consisting of: a vacuum, tube, a non-vacuum tube, a plastic tube, a glass tube, a rigid tube, a non-rigid tube, a semi rigid tube and any combination thereof.

It is another object of the present invention to provide the system as defined above, wherein the vacuum tube is adapted such that, when containing the whole blood (5), and centrifuged after treatment, yields separation fractions comprising, a first fraction of RBCs sediment and granulocytes (6), a second fraction of the gel (3), a third fraction comprising platelets (7), and fourth fraction of PPP (8)

It is another object of the present invention to provide the system as defined above, wherein the collection syringe for collecting platelets poor plasma (PPP) is adapted for discarding PPP.

It is another object of the present invention to provide the system as defined above, wherein the platelets rich plasma (PRP) collection syringe is adapted for drawing the remaining PRP after re-suspending of the PRP portion.

It is another object of the present invention to provide the system as defined above, further comprising a stopcock adapted as a turning plug which controls the flow of fluid from the PPP collection syringe to the blood collection tube.

It is another object of the present invention to provide the system as defined above, further comprising a stopcock adapted as a turning plug which controls the flow of fluid from the PRP portion collection syringe to the blood collection tube.

It is another object of the present invention to provide the system as defined above, wherein the anticoagulant is selected from the group consisting of: buffer citrate, ACD, modified ACD (citric/citrate dextrose), heparinate salts, EDTA salts, iodo acetate salts, oxalate salts, fluoride salts as water solutions or lyophilized material or wet or dry spray on inner wall and so forth and any combination thereof.

It is another object of the present invention to provide the system as defined above, wherein the anticoagulant comprises any type of anticoagulant designed for preventing clotting of blood.

It is another object of the present invention to provide the system as defined above, wherein the separation gel is at density of 1.04-1.09 g/cm³.

It is another object of the present invention to provide the system as defined above, wherein the centrifugation is performed for about 10-30 min.

It is another object of the present invention to provide the system as defined above, wherein the PPP is discardable up to 90% of plasma from the blood collection tube.

It is another object of the present invention to provide the system as defined above, wherein the PPP is discardable at least 50% of plasma from the blood collection tube.

It is another object of the present invention to provide the system as defined above, wherein the PPP is discarded about 90% of plasma from the blood collection tube.

It is another object of the present invention to provide the system as defined above, wherein the collection syringe for collecting platelet rich plasma (PRP) contains additives selected from the group consisting of: Hyaluronic acid (HA), collagen, allograft bone, autograft bone, bone substitutes, autologous adults stem cells, CaCl₂, thrombin, collagen and any combination thereof.

It is another object of the present invention to provide the system as defined above, wherein the collection syringe for collecting platelets rich plasma (PRP) is adapted for a mixture of the obtained platelet rich plasma (PRP) with an activating agent selected from the group consisting of Thrombin CaCl₂, collagen and ADP.

It is one object of the present invention to provide a method for producing of platelet rich plasma (PRP), the method comprising steps of:

(a) obtaining a closable system for preparing platelet rich plasma (PRP) comprising: (i) a collection tube for collecting blood (1), (ii) at least one stopper (2); (iii) an anticoagulant (4), (iv) a separation gel (3), (v) a collection syringe for collecting platelets poor plasma (PPP) (10), (vi) a collection syringe for collecting platelets rich plasma (PRP) portion (11), and (vi) at least one needle for attaching to each syringe, and

(b) placing the anticoagulant portion and the portion of separation gel in the tube; (c) filling the collection tube for collecting blood with whole blood sample, (d) separating by centrifugation, thereby obtaining separation fractions comprising, a first fraction of RBCs sediment and granulocytes, a second fraction of the gel, a third fraction of platelets, and fourth fraction of PPP solution, (e) discarding a portion of PPP with the first collection syringe combined with the needle; (f) re-suspending of the platelets with other separated blood components, (g) extracting the remaining PRP and portion of WBCs solution with the PRP collection syringe; such that a WBC-selective PRP is obtained.

It is one object of the present invention to provide a method for producing of enriched stem cells sample, the method comprising steps of:

(a) obtaining a closable system for obtaining a cellular fraction enriched with defined cells effective in promoting a defined cellular response from an in vivo cellular sample comprising: (i) a collection tube for collecting a cell suspension (1), (ii) at least one stopper (2); (iii) an anticoagulant portion (4), (iv) a portion of separation gel (3), (v) a first collection syringe for collecting a cellular sample, (vi) a second collection syringe for collecting the enriched cellular portion; and, (vii) at least one needle (10) for attaching to each syringe, and

(b) placing the anticoagulant portion and the portion of separation gel in the tube,

(c) filling cell suspension with cell culture sample, (d) separating by centrifugation, thereby obtaining separation fractions comprising, a first fraction of cell suspension, a second fraction of the gel, a third fraction of enriched cellular portion, and fourth fraction of poor cellular fluid, (e) discarding a portion of poor cellular fluid with the first collection syringe combined with the needle, (f) re-suspending of the cellular fluid with other separated cell components, and (g) extracting the remaining enriched cellular portion with the enriched cellular portion collection syringe such that an enriched selective stem cells fraction is obtained.

It is another object of the present invention to provide the method as defined above, wherein the third fraction is re-suspended to obtain PRP.

It is another object of the present invention to provide the method as defined above, wherein the step of extracting the remaining PRP and portion of WBCs solution is with PRP collection syringe attached to a filter unit.

It is another object of the present invention to provide the method as defined above, wherein the step of extracting the remaining enriched cellular portion with the enriched cellular portion collection syringe is with a filter unit.

It is another object of the present invention to provide the method as defined above, wherein the discarding up to 90% of PPP from the blood collection tube.

It is another object of the present invention to provide the method as defined above, wherein the discarding about 90% of PPP from the blood collection tube.

It is another object of the present invention to provide the method as defined above, wherein the anticoagulant comprises any type of anticoagulant designed for preventing clotting of blood.

It is another object of the present invention to provide the method as defined above, wherein the enriched cellular portion collection syringe contains additives selected from the group consisting of: Hyaluronic acid (HA), collagen, allograft bone, auto graft bone, bone substitutes, autologous adults stem cells, CaCl₂, thrombin, collagen and any combination thereof.

It is another object of the present invention to provide the method as defined above, wherein the enriched cellular fraction is optimally transferred using the second collection syringe to a vessel containing additives selected from the group consisting of: Hyaluronic acid (HA), collagen, allograft bone, auto graft bone, bone substitutes, autologous adults stem cells, CaCl₂, thrombin, collagen and any combination thereof.

It is another object of the present invention to provide the method as defined above, wherein the PRP collection syringe comprises at least two split barrels, whilst one of the barrels contains activators or/and additives and another barrel serves for PRP collection. It is another object of the present invention to provide the method as defined above, further comprising the step of mixing the concentrated PRP or stem cell portion with hemostat or biodegradable scaffolds

It is another object of the present invention to provide the method as defined above, further comprising the step of mixing the obtained platelet rich plasma (PRP) with an activating agent selected from the group consisting of Thrombin, CaCl₂, collagen and ADP.

It is another object of the present invention to provide the method as defined above, wherein the filter unit is of 3-50 μm.

It is another object of the present invention to provide the method as defined above, wherein the tube is selected from the group consisting of: a vacuum tube, a non-vacuum tube, a plastic tube, a glass tube, a rigid tube, a non rigid tube, a semi rigid tube and any combination thereof.

It is another object of the present invention to provide the method as defined above, wherein the separation gel is at density of 1.04 to about 1.09 g/cm³.

It is another object of the present invention to provide the method as defined above, including discarding up to 90% of PPP from the blood collection vacuum tube.

It is another object of the present invention to provide the method as defined above, including discarding about 90% of PPP from the blood collection vacuum tube.

It is another object of the present invention to provide the method as defined above, wherein the anticoagulant is selected from the group consisting of: buffer citrate, ACD, modified ACD (citric/citrate dextrose), heparinate salts, EDTA salts, iodo acetate salts, oxalate salts, fluoride salts as water solutions or lyophilized material or wet or dry spray on inner wall and so forth and any combination thereof.

It is another object of the present invention to provide the method as defined above, wherein the anticoagulant comprises any type of anticoagulant designed for preventing clotting of blood.

It is another object of the present invention to provide the method as defined above, wherein the platelets rich plasma (PRP) collection syringe includes additives selected from the group consisting of: Hyaluronic acid (HA), collagen, allograft, auto graft bone, bone substitutes, autologous adults stem cells, CaCl₂, thrombin, collagen and any combination thereof.

It is another object of the present invention to provide the method as defined above, further comprising the step of mixing the obtained platelet rich plasma (PRP) with an activating agent selected from the group consisting of Thrombin, CaCl₂, collagen and ADP.

BRIEF DESCRIPTION OF THE INVENTION

In order to understand the invention and to see how it may be implemented in practice, a few preferred embodiments will now be described, by way of non-limiting example only, with reference to be accompanying drawings, in which:

FIG. 1 shows a closed system for derivation of WBC-selective PRP, in accordance with a preferred embodiment of the present invention;

FIG. 2 illustrates a flow chart of a method for producing a WBC-selective PRP in a closed system, in accordance with a preferred embodiment of the present invention; and,

FIG. 3 illustrates a flow chart of a method for producing an enriched cellular portion in a closed system, in accordance with a preferred embodiment of the present invention;

DETAILED DESCRIPTION OF EMBODIMENTS

The following description is provided so as to enable any person skilled in the art to make use of the invention and sets forth the best modes contemplated by the inventor of carrying out this invention. Various modifications, however, will remain apparent to those skilled in the art, since the generic principles of the present invention have been defined specifically to provide methods and systems for producing high levels of platelet rich plasma (PRP).

The term “PRP” or “platelet rich plasma” as used herein should be understood to mean a blood-derived product which comprises platelets concentrated in a small volume of plasma.

The term “WBC-selective PRP” as used herein should be understood to encompass PRP having reduced number of white blood cells or a selective population of WBCs. The term “WBC-selective PRP” typically refers to a PRP that comprises WBCs at a concentration which is less than 50%, at times less than 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2% or even less than 1% than that in the blood. The term “WBC-selective PRP” may also refer to PRP devoid of a certain population of WBCs, typically such that are filtered-out by a filter with a certain pore size.

The PRP obtained in accordance with the invention may be used for treating aging skin and wrinkled-skin cells, accelerating healing of bone cells, treating surgery wounds, diabetic wounds, ulcer wounds, and pressure wounds and is used as autologous biological cells formation and/or biological glue.

In particular, some exemplary embodiments of the invention enable the preparation of PRP in a practitioner's treatment settings. The preparation of a WBC-selective PRP according to the invention is relatively rapid, e.g. about 15, 30, 40, 50 or 60 minutes.

The term “Stem cell” as used herein should be understood to mean biological cells or vehicle found in all multicellular organisms. Accessible sources of autologous adult stem cells are Bone marrow, Adipose tissue (lipid cells), umbilical cord blood and hematopoetic blood.

In accordance with the preferred embodiment of the present invention all the elements of the invention, such as tubes, syringes etc., are combined in a closed system. The closed system eliminates direct contact with the fluid path, while helping to reduce the danger of contamination and infection. It is known in the art that whole blood components collected in a non-sterile, or “open”, system (i.e. one that is open to communication with the atmosphere) must, under governmental regulations, be transfused within twenty-four hours. However, when cell suspensions such as bone marrow, adipose stem cells or whole blood components are collected in a sterile, or “closed”, system (i.e., one that is closed to communication with the atmosphere), the cell suspension or red blood cells can be stored up to forty-two days (depending upon the type of anticoagulant and storage medium used); the platelet concentrate can be stored up to five days (depending upon the type of storage container); and the platelet-poor plasma may be frozen and stored for even longer periods. The apparatus and methods that embody the features of the invention are particularly well suited for use in association with closed blood collection systems and conventional sterile connection techniques, thereby permitting separation to occur in a sterile, closed environment.

The present invention provides systems and methods for separating platelets from peripheral blood or from tissue derived cellular samples, such as bone marrow, umbilical cord blood, blood or adipose stem cells respectively.

The present invention presents a system for obtaining a cellular fraction enriched with defined cells, which have been reported to be effective in promoting a defined cellular response from an in vivo cellular sample comprising: (a) a collection tube for collecting a cell suspension, (b) at least one stopper, (c) an anticoagulant portion, (d) a portion of separation gel, (e) a first collection syringe for collecting a plasma or cellular sample, (f) a second collection syringe for collecting the enriched cellular portion and, (g) at least one needle (10) for attaching to at least one of the syringes.

The system is closable to the atmosphere. The cell suspension collection tube contains the gel and the anticoagulant and blood or cell suspension sample or cell culture. The tube containing cell suspension is adapted such that, when centrifuged after treatment yields separation fractions, a first fraction comprising cellular fluid, a second fraction comprises gel layer, a third fraction comprises enriched cellular portion on top of the gel layer and a fourth fraction comprises the cell poor portion.

The system is further adapted such that the enriched cellular portion is harvested with a collection syringe with a needle for collecting cellular samples.

Furthermore, the collecting of an enriched cellular portion with the needle is dimensionally configured for extraction of cells remaining after re-suspending of the enriched cellular portion. Isolation of stem cells may be performed whether from somatic tissues, or whole blood. Protocols may further be adjusted based on the stem cell source or species.

The present invention further provides a method of direct aspiration into a vacuum bone marrow collection tube using a suitable needle that penetrates the bone. BM aspirate should be mixed with anticoagulant and processed for mononuclear cells separation.

The present invention further provides a system which includes a centrifugation to obtain PRP or BM-/adipose/umbilical cord blood derived mesenchymal stem cells (MSC) or Hematopoietic stem cells. The Hematopoietic and MSC are nucleated cells, whilst platelets are anucleated cytoplasmic fragments of megakaryocyes. Therefore, different separation gel having different density is applied for PRP and MSC separation. The concentrated solution of BMSC comprises different progenitor cells such as mesenchymal stem cells (MSC), hematopoietic stem cells (HSCs), endothelial progenitor stem cells (EPCs). These cells as well as PRP, promote cellular responses and tissue repair by secreting trophic, paracrine, and growth factors which play a key role in the wound-healing process by initiating and maintaining cell proliferation, angiogenesis, matrix deposition and the like.

In another embodiment of the present invention, Hematopoietic and MSCs have the ability to differentiate, and they help to establish new vascular and musculoskeletal tissues. Furthermore, concentrated hematopoietic stem cells or mesenchymal stem cells may be used to treat certain hematological malignancies.

In another embodiment of the present invention, a possible implementation of PRP or MSC treatment for bone-healing purposes, is the bone graft (i.e., autograft, allograft, or synthetic) which may be soaked in PRP or mixed/infused with MSC. Furthermore, PRP and/or stem cell concentrate may be mixed with hemostat or biodegradable scaffolds for internal or external (for example wounds, dentistry) use. Human adipose tissue obtained by liposuction, can be processed to obtain a population of cells such as processed lipoaspirate (PLA). This tissue, in addition to adipogenic, endothelial and vascular progenitor cells, also contains multipotent cell types, termed adipose stem cells (ADSCs), which have the ability to undergo self-renewal and multilineage differentiation to bone, cartilage and muscle, as well as fat, similarly to their bone marrow-derived counterparts. The use of autologous ASCs as cellular regeneration therapy is both safe and efficacious in preclinical and clinical studies of injury and disease.

Without wishing to be bound by theory, Platelet-rich plasma can enhance the proliferation of human ADSCs. Furthermore, peripheral blood may be processed to obtain concentrated stem cell portion. Peripheral blood contains circulating hematopoietic stem cells and their concentration in the blood, can be markedly elevated by administration of known substances, such as granulocyte colony-stimulating factor (G-CSF), which is used in leukopheresis techniques to increase significantly the number of stem cell progenitors in the circulation by inducing. This is being referred to as mobilized peripheral blood.

In another embodiment of the present invention, umbilical cord blood is a source of hematopoietic stem (HSC) and progenitor cells for the treatment of a variety of malignant and non-malignant disorders. The system described herein can be used to obtain stem cell concentrate from cord blood for cryopreservation and treatment.

Reference is herein made to FIG. 1 which illustrates a closable system for obtaining platelet rich plasma (PRP) in whole blood sample.

A closable system for producing PRP comprises: (a) a blood collection tube 1, (b) at least one stopper 2, (c) an anticoagulant portion 4, (d) a portion of separation gel 3, (e) a platelets poor plasma (PPP) collection syringe 10 including sharp long needle 10 a, and (f) a platelets rich plasma (PRP) collection syringe 11 including sharp long needle 11 a.

In some embodiments, the test tube 1 is provided with a stopper, a cover or any closer 2, typically made of rubber.

In some embodiments, test tubes are made of glass or MPA (modified poly Amide) or modified PET (Poly Ethylene Terephthalate). In some embodiments, the test tube has a layered structure such that the interior wall of the test tube comprises polypropylene. Test tubes can further be provided with a special stopper. By way of a non-limiting example the cover is made of butyl rubber or its halo derivative formulations at hardness between 40-60 Shore A. The hardness assures stable vacuum for at least the shelf life of the test tube which can be between 18-24 months. The tube can be vacuum or non-vacuum. For non-vacuum tubes, blood is drawn by standard means, such as a needle and syringe and transferred into the tube through a security opening, which sustains closed system environment.

The test tubes used can be of various sizes which depend on the required quantity of whole blood/cellular fluid to be drawn from the treated subject. The test tubes have typically a size suitable for blood samples in the range of 4 ml to 100 ml.

The collection tube is selected from the group consisting of: vacuum, a non vacuum tube, a rigid tube, a non-rigid tube, a semi-rigid tube, plastic tube, glass tube, and any combination thereof.

In accordance with the preferred embodiment of the present invention, FIG. 1 shows the vacuum tube 1 consisting of a separation gel 3 layer at a density of 1.04-1.09 g/cm³ and an anticoagulant layer 4. The anticoagulant 4 is selected from the group consisting of: such as, but not limited to buffer citrate, ACD, modified ACD (citric/citrate dextrose), heparinate salts, EDTA salts, iodo acetate salts, oxalate salts, fluoride salts as water solutions or lyophilized material or wet or dry spray on inner wall and so forth, or any type of anticoagulant designed for preventing clotting of blood.

In some embodiments, test tubes contain a barrier-forming substance to assist in the centrifugation-based separation of blood into its components. Such barrier-forming substance may be of the kind known per se, for example gel polymers. Gel polymers may, for example, be based on polyacrylic, polyolefin or polyester with a specific gravity in the range of about 1.04 to 1.09 g/cm³.

In a first step of the procedure, whole blood sample 5 withdrawn from a subject is inserted into a blood collection tube which comprises the gel layer 3 and the anticoagulant layer 4. In order to perform a separation of whole blood 5 a centrifugation procedure 110 is performed on the basis of specific gravity at 300-2000 g for about 10 to about 30 minutes, resulting in four fractions. Following centrifugation 110, blood components, particularly red blood cells and most of the granulocytes 6, will remain beneath the gel barrier 3 and other components, including platelets, will remain above it. The first and heaviest fraction at the bottom of the tube comprises red blood cells (RBCs) and most of the granulocytes layer 6 as a sediment layer, the second fraction comprises the gel layer 3, the third fraction, on the top of the gel, comprises platelet rich plasma (PRP) as a thin layer 7, and the last (fourth) fraction comprises platelet poor plasma (PPP) solution 8 as a supernatant layer. The system remains closed in all of the steps of the procedure and the blood does not coagulate or become contaminated.

In yet another embodiment of the invention, while test tube may optionally include an anti-coagulant, in some procedures an anti-coagulant may a priori not be included. For example, if the blood sample is withdrawn and maintained in cold conditions, an anti-coagulant may not be needed. In addition, in some procedures, the anti-coagulant may be mixed with the whole blood which was withdrawn from a subject, prior to inserting the whole blood into the tube.

Through agitation/mixing, it can then mix with the anticoagulant, optionally included within the test tube. Alternatively, the blood sample may be mixed with an anti-coagulant, prior to insertion into tube. The test tube is then subjected to centrifugation. Centrifugation is used to separate the red blood cells (RBCs) and most of the granulocytes from the plasma and the platelets.

Centrifugation may, for example, be at about 300-2000 g and for a time period of 10-30 minutes. Centrifugation results in platelet enrichment 2-10 times compared to their native concentration in whole blood.

In yet another embodiment of the invention, anticoagulant will be used, the blood collection tube containing separation gel layer and whole blood sample will be centrifuged, resulting in four fractions: the first and heaviest fraction at the bottom of the tube comprises red blood cells (RBCs) and most of the granulocytes layer 6 as a sediment layer, the second fraction comprises the gel layer 3 as a barrier between the RBCs fraction and a plasma fraction, the third fraction comprises PRP 7, on the top of the gel, and the last fraction comprises PPP solution 8. After centrifugation 110 a PPP collection syringe 10 with a sharp long needle 10 a is inserted into the blood collection tube 120 and a portion of PPP 8 is removed 130 from the tube. The platelets are concentrated at the bottom end of the plasma fraction, typically forming a pellet. Typically, more than half of the plasma is PPP, therefore removal of more than half of the upper phase in the blood collection tube is required. By removal of part of the plasma to yield more enriched plasma and by subsequent agitation, a PRP fraction 7 is obtained.

In some embodiments of the invention the amount of PPP 8 which extracted from the blood collection tube 1 is up to 90% of PPP. In another embodiment of the invention, the amount of PPP 8 which extracted from the tube 1 is at least 90% of PPP. In yet another embodiment of the invention the amount of PPP which extracted from the tube 1 is about 90% of PPP.

Extraction of the PPP from the tube is performed using a collection syringe with optional 1-3 way stopcock 11 c attached to a long sharp needle 11 a.

In another embodiment of the invention, the PRP collection syringe 11 may also include a filter unit 11 b having an effective pore size of about 3-50 μm such to permit passage of platelets and not permit passage of white blood cells (WBCs).

Re-suspending of the platelets with optional separated blood components 160 such as WBCs and growth factors is performed via repeated inversion of the tube. The collection syringe can be attached to an optional stopcock 11 as a valve or turning plug which controls the flow of fluid from the PPP collection syringe to the blood collection tube. The collection syringe can be also attached to an optional filter unit (with or without a stopcock unit). The PRP collection syringe may be either pre-washed or un-washed, with activator such as Thrombin, CaCl₂, collagen solution, ADP or silicate solution or suspension. The PRP collection syringe may comprise two or more split barrels, when one of the barrels may contain activators or/and additives and another barrel can collect PRP. Alternatively, PRP may be transferred by the PRP collection syringe to another vessel containing activators and/or additives.

The remaining amount of the fraction comprising; PRP 7 which can be combined with WBCs, is drawn 170 using a PRP portion collection syringe 11, attached to the hub of the long sharp needle 11 a or via attaching a syringe to another port of the stopcock 11 c with an optional filter unit 11 b.

In another embodiment of the invention, an optional stopcock 11 as a valve or turning plug controls the flow of fluid from the PPP collection syringe to the blood collection tube. In another embodiment of the invention a two or three-way stopcock can be used also to turn off the flow of one solution and turn on the flow of another. The PRP portion collection syringe 11 are transferred.

In another embodiment of the invention, using the method as described above, the platelets rich plasma (PRP) collection syringe may contain an additive selected from the group consisting of: Hyaluronic acid (HA), collagen, bone substitutes, allograft or autograft bone, previously derived autologous adult stem cells, CaCl₂, thrombin of human recombinant or animal origin, collagen, and any combination thereof. Furthermore, the PRP fraction that was collected may be transferred by a PRP collection syringe to another vessel which contains the aforementioned additives. The PRP collection syringe may comprise two or more split barrels, when one of the barrels may contain activators or/and additives and another barrel serves for PRP collection.

Furthermore, the PRP or stem cell concentrate may be mixed with hemostat or biodegradable scaffolds for internal or external (wounds, dentistry, for example) use.

Reference is herein made to FIG. 2 which illustrates a procedure for preparing selective PRP. Whole blood sample can be drawn from a subject by using a conventional blood drawing device 200, having a vein needle, a flexible tube and separation tube adapter with a piercing member (holder attached to Luer adapter attached to PSV Butterfly). By piercing the central portion of the stopper and as a consequence of the vacuum inside the test tube, blood is withdrawn there into. Separation Gel and anticoagulant, optionally included within the test tube, becomes mixed with the blood sample inhibiting clotting. Eventually, after disconnecting the tube from the blood drawing device, a test tube with a blood sample, optionally mixed with an anticoagulant is obtained.

In other embodiments of the present invention, the blood can be drawn from a subject by using a conventional blood drawing device, such as PSV butterfly and syringe and transferred from the syringe into a non-vacuum tube.

In some exemplary embodiments of the invention, there is provided a method for producing of platelets rich plasma (PRP) in a closable system, the method comprising steps of: (a) obtaining a closed system for preparing platelet rich plasma (PRP) 200 comprising: (i) a blood collection tube, (ii) an anticoagulant portion, (iii) a portion of separation gel, (iv) a platelets poor plasma (PPP) collection syringe including sharp long needle; and (v) a platelets rich plasma (PRP) collection syringe including sharp long needle and a filter unit;

(b) placing the anticoagulant portion and the portion of separation gel in the tube 210, (c) filling the blood collection tube with whole blood sample 220, (d) separating by centrifugation thereby obtaining separation fractions comprising, a first fraction of RBCs with some granulocytes sediment, a second fraction of the gel, a third fraction of PRP, and fourth fraction of PPP solution 230, (e) discarding a portion of PPP with the PPP collection syringe comprising a sharp needle 240, (f) re-suspending of the platelets with other separated blood components 250, (g) extracting the remaining PRP and portion of WBCs solution with the PRP collection syringe 260 which may include a filter unit, such that a WBC-selective PRP is obtained.

In another embodiment of the invention the method as described above, comprises a filter unit of 3 to about 50 μm.

In another embodiment of the invention the method as described above wherein the tube is selected from the group consisting of: a vacuum, a non-vacuum tube, a rigid tube, a non-rigid tube, a semi rigid tube, a plastic tube, a glass tube, and any combination thereof.

In another embodiment of the invention the method as described above wherein the separation gel is at density of 1.04 to about 1.09 g/cm³.

In another embodiment of the invention the method as described above wherein the discarding up to 90% of PPP from the blood collection vacuum tube.

In another embodiment of the invention the method as described above wherein the discarding about 90% of PPP from the blood collection vacuum tube.

In another embodiment of the invention the method as described above, wherein the anticoagulant is selected from the group consisting of: buffer citrate, ACD, modified ACD (citric/citrate dextrose), heparinate salts, EDTA salts, iodo acetate salts, oxalate salts, fluoride salts as water solutions or lyophilized material or wet or dry spray on inner wall and so forth and any combination thereof.

In another embodiment of the invention the method as described above, wherein the anticoagulant comprises any type of anticoagulant designed for preventing clotting of blood.

In another embodiment of the invention the method as described above, wherein the platelets rich plasma (PRP) collection syringe may contain additives selected from the group consisting of: Hyaluronic acid (HA), collagen, bone substitutes, allograft or autograft bone, previously derived autologous adult stem cells, CaCl₂, thrombin of human recombinant or animal origin, collagen and any combination thereof. The PRP collection syringe may comprise at least two split barrels, whilst one of the barrels may contain activators or/and additives and the another barrel is used for PRP collection. The PRP portion which was collected using the PRP collection syringe, may be transferred to another vessel which contains the aforementioned additives. Furthermore, the concentrated PRP or the stem cell fraction may be mixed with hemostat or biodegradable scaffolds for internal or external use (e.g. wounds, dentistry).

Reference is herein made to FIG. 3 which illustrates a method for producing of enriched stem cells sample, the method comprising steps of:

(a) obtaining a closable system for obtaining a cellular fraction enriched with defined cells effective in promoting a defined cellular response from an in vivo cellular sample, 300 comprising: (i) a collection tube for collecting a cell suspension (1); (ii) at least one stopper, (iii) an anticoagulant portion, (iv) a portion of separation gel, (v) a first collection syringe for collecting a cellular sample, (vi) a second collection syringe for collecting the enriched cellular portion and, (vii) at least one needle (10) for attaching to each the syringe. (b) placing the anticoagulant portion and the portion of separation gel in the tube 310, (c) filling cell suspension with cell culture sample 320, (d) separating by centrifugation there by obtaining separation fractions comprising, a first fraction of cell suspension, a second fraction of the gel, a third fraction of enriched cellular portion, and fourth fraction of poor cellular fluid 330, (e) discarding a portion of poor cellular fluid with the first collection syringe combined with the needle 340, (f) re-suspending of the cellular fluid with other separated cell components, (g) extracting the remaining enriched cellular portion with the enriched cellular portion collection syringe, optionally combined with a filter unit 350, such that an enriched selective stem cells fraction is obtained.

The system is adapted such that the cell poor portion is discardable with the first collection syringe with the needle. The second collection syringe with the needle are dimensionally configured for extraction of cells remaining after re-suspending of the enriched cellular portion. The term cell suspension is a peripheral blood sample or a stem cell sample containing cells such as bone marrow or adipose or umbilical cord blood stem cells.

In another embodiment of the present invention, a system for obtaining an enriched mesenchymal stem cell (MSC) or hematopoetic stem cells portion may be adapted, the system comprising:

(a) a collection tube for collecting cell suspension, (b) at least one stopper, (c) an anticoagulant portion, (d) a portion of separation gel, (e) a first collection syringe for collecting poor cellular fluid, (f) a second collection syringe for collecting enriched mesenchymal stem cell (MSC) portion (11) and (g) at least one needle (10) for attaching to each syringe.

The system is closable to the atmosphere. The collection tube comprising the gel and the anticoagulant. The collection tube containing cell suspension is adapted such that, when containing the cell suspension and centrifuged after treatment yields separation fractions, a first fraction comprises red blood cells (RBCs), a second fraction comprises gel layer, a third fraction comprises cellular layer containing MSC or hematopoietic stem cells on top of the gel layer and a fourth fraction comprises poor cellular fluid.

The system is adapted such that the poor cellular fluid is discardable with the first collection syringe.

The second collection syringe with the needle are dimensionally configured for extraction of the enriched MSC portion remaining after re-suspending of the MSC portion.

The invention further provides a system for obtaining an enriched bone marrow fraction may be adapted, the system comprising: (a) a collection tube for collecting bone marrow, (b) at least one stopper, (c) an anticoagulant portion, (d) a portion of separation gel, (e) a first collection syringe for collecting fluid poor in cells, (f) a second collection syringe for collecting a portion enriched in bone marrow cells, and (g) at least one needle for attaching to each syringe.

The system is closable to the atmosphere. The collection tube for collecting bone marrow contains the gel and the anticoagulant; the tube containing the bone marrow is adapted such that when centrifuged after treatment yields separation fractions, a first fraction comprises primarily red blood cells (RBCs) sediment, a second fraction comprises gel layer, a third fraction comprises cellular fraction of enriched bone marrow cells on top of the gel layer and a fourth fraction comprises poor cellular fluid.

The poor cellular fluid is discarded using the first collection syringe.

The second collection syringe with the needle are dimensionally configured for extraction of the enriched bone marrow cells after re-suspending of the remaining bone marrow cells portion.

The system further comprises means for treating cell aspirate before the latter is applied into the collection tube.

The invention further provides a system for obtaining a low medium density fraction enriched with defined cells effective in promoting a defined cellular response from an in vivo cellular sample comprising: (a) a collection tube for collecting a cell suspension, (b) an anticoagulant portion to prevent clotting of cells, (c) a portion of separation gel, (d) a first collection syringe for collecting a cell depleted fluid; (e) a second collection syringe for collecting the enriched cellular portion; and, (d) at least one needle for attaching to each syringe.

The system is closable to the atmosphere. The cell suspension collection tube contains the gel and the anticoagulant. The tube is adapted such that, when containing cell suspension and centrifuged after treatment yields fractions of different densities, a first high density fraction, a second medium-high density fraction (3), a third medium-low density fraction on top of the gel layer and a fourth fraction low density fraction, the third medium-low density fraction substantially containing enriched defined cells; the enriched defined cells selected from the group consisting of enriched bone marrow stem cells, enriched MSC or platelet rich plasma (PRP).

In the foregoing description, embodiments of the invention, including preferred embodiments, have been presented for the purpose of illustration and description.

They are not intended to be exhaustive or to limit the invention to the precise form disclosed. Obvious modifications or variations are possible in light of the above teachings. The embodiments were chosen and described to provide the best illustration of the principals of the invention and its practical application, and to enable one of ordinary skill in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. All such modifications and variations are within the scope of the invention as determined by the appended claims when interpreted in accordance with the breadth they are fairly, legally, and equitably entitled. 

What is claimed is:
 1. A system closable to the atmosphere for obtaining a cellular fraction enriched in stem cells comprising: a collection tube containing an anticoagulant portion and a separation gel with a density of 1.04 to 1.09 g/cm³ and having at least one stopper for maintaining said collection tube closed to the atmosphere; a first collection syringe for collecting fluid depleted in stem cells from said collection tube after centrifugation; a second collection syringe for collecting said cellular fraction enriched in stem cells from said collection tube after centrifugation, said second collection syringe further comprising a filter adapted to filter cells included in said cellular fraction; and at least one needle for attaching to each said syringe and adapted to be received in the collection tube while said collection tube remains closed to the atmosphere; wherein the collection tube is adapted to yield, after centrifugation, in order, a first fraction containing primarily red blood cells; a second fraction containing the gel; a third fraction enriched in stem cells; and a fourth fraction depleted in stem cells.
 2. The system according to claim 1, comprising first and second needles adapted to be attached to said first and second collection syringes respectively.
 3. The system according to claim 1, wherein the filter in said second collection syringe is adapted to selectively prevent passage of white blood cells into said cellular fraction enriched in stem cells.
 4. The system according to claim 3, wherein the filter unit has a pore size of 3 to 50 μm.
 5. A system closable to the atmosphere for obtaining plasma enriched in platelets, comprising: a collection tube containing an anticoagulant portion and a separation gel with a density of 1.04 to 1.09 gr/cm³ and having at least one stopper for maintaining said collection tube closed to the atmosphere; a first collection syringe adapted to collect a portion of fluid relatively depleted in platelets from said collection tube after centrifugation; a second collection syringe adapted to collect said plasma enriched in platelets from said collection tube after centrifugation, said second collection syringe further comprising a filter unit adapted to filter cells included in said plasma enriched in platelets; and at least one needle for attaching to each said syringe and adapted to be received in the collection tube while said collection tube remains closed to the atmosphere; wherein, the collection tube is adapted to yield, after centrifugation, a first fraction containing primarily red blood cells on a first side of the gel, and a second fraction comprising stem cells, platelets and plasma on a side of the gel opposite said first side.
 6. The system according to claim 5, comprising first and second needles adapted to be attached to said first and second collection syringes respectively.
 7. The system according to claim 5, wherein the filter unit in said second collection syringe is adapted to selectively prevent passage of white blood cells into said cellular fraction enriched in stem cells.
 8. The system according to claim 5, wherein the filter unit has a pore size in a range of 3-50 μm.
 9. The system according to claim 5, wherein the separation gel is a barrier-forming gel polymer based on polacrylic, polyolefin or polyester.
 10. The system according to claim 9, wherein the barrier-forming separation gel is based on polyolefin.
 11. A method for obtaining plasma enriched in platelets, comprising: filling a collection tube containing an anticoagulant and a gel polymer based on polyolefin with a specific gravity in a range of 1.04 to 1.09 g/cm³, and having at least one stopper for maintaining said collection tube closed to the atmosphere, with whole blood; separating the whole blood in the collection tube by centrifugation to obtain a first fraction of red blood cells, sediment and granulocytes; a second fraction of the gel; a third fraction of platelets; and a fourth fraction of plasma depleted in platelets; collecting with a first collection syringe a portion of the fraction of plasma depleted in platelets; discarding the portion collected with the first collection syringe; resuspending the third fraction of platelets; and extracting the plasma enriched in platelets with a second collection syringe while said collection tube remains closed to the atmosphere; wherein said plasma enriched in platelets contains white blood cells at a concentration less than 50% of the concentration in the whole blood.
 12. The method according to claim 11, comprising filtering white blood cells from said plasma enriched in platelets with a filter during said extracting.
 13. The method according to claim 12, wherein said filter has a pore size between 3-50 μm.
 14. The method according to claim 11, wherein said separating comprises centrifugation at about 300-2000 g for 10 to 30 minutes.
 15. The method according to claim 15, wherein said centrifugation results in platelet enrichment in said platelet enriched plasma of 2 to 10 times compared to said whole blood. 